SummaryImmunodeficient non-obese diabetic (NOD)-severe combined immunedeficient (scid) mice bearing a targeted mutation in the gene encoding the interleukin (IL)-2 receptor gamma chain gene (IL2rg null ) engraft readily with human peripheral blood mononuclear cells (PBMC). Here, we report a robust model of xenogeneic graft-versus-host-like disease (GVHD) based on intravenous injection of human PBMC into 2 Gy conditioned NOD-scid IL2rg null mice. These mice develop xenogeneic GVHD consistently (100%) following injection of as few as 5 ¥ 10 6 PBMC, regardless of the PBMC donor used. As in human disease, the development of xenogeneic GVHD is highly dependent on expression of host major histocompatibility complex class I and class II molecules and is associated with severely depressed haematopoiesis. Interrupting the tumour necrosis factor-a signalling cascade with etanercept, a therapeutic drug in clinical trials for the treatment of human GVHD, delays the onset and progression of disease. This model now provides the opportunity to investigate in vivo mechanisms of xenogeneic GVHD as well as to assess the efficacy of therapeutic agents rapidly.

SummaryImmunodeficient hosts engrafted with human lymphohaematopoietic cells hold great promise as a preclinical bridge for understanding human haematopoiesis and immunity. We now describe a new immunodeficient radioresistant non-obese diabetic mice (NOD) stock based on targeted mutations in the recombination activating gene-1 (Rag1 null ) and interleukin (IL)-2 receptor common gamma chain (IL2rg null ), and compare its ability to support lymphohaematopoietic cell engraftment with that achieved in radiosensitive NOD.CB17-Prkdc scid (NOD-Prkdc scid ) IL2rg null mice. We observed that immunodeficient NOD-Rag1 null IL2rg null mice tolerated much higher levels of irradiation conditioning than did NOD-Prkdc scid IL2rg null mice. High levels of human cord blood stem cell engraftment were observed in both stocks of irradiation-conditioned adult mice, leading to multi-lineage haematopoietic cell populations and a complete repertoire of human immune cells, including human T cells. Human peripheral blood mononuclear cells also engrafted at high levels in unconditioned adult mice of each stock. These data document that Rag1 null and scid stocks of immunodeficient NOD mice harbouring the IL2rg null mutation support similar levels of human lymphohaematopoietic cell engraftment. NOD-Rag1 null IL2rg null mice will be an important new model for human lymphohaematopoietic cell engraftment studies that require radioresistant hosts.

The major obstacle to successful discordant xenotransplantation is the phenomenon of hyperacute rejection (HAR). In the pig-to-primate discordant transplant setting, HAR results from the deposition of high-titre anti-alpha-galactosyl antibodies and complement activation leading to endothelial cell destruction and rapid graft failure. To overcome HAR, we developed an enzymatic carbohydrate remodelling strategy designed to replace expression of the Gal alpha-1,3-Gal xenoepitope on the surface of porcine cells with the non-antigenic universal donor human blood group O antigen, the alpha-1,2-fucosyl lactosamine moiety (H-epitope). Xenogenic cells expressing the human alpha-1,2-fucosyltransferase expressed high levels of the H-epitope and significantly reduced Gal alpha-1,3-Gal expression. As a result, these cells were shown to be resistant to human natural antibody binding and complement-mediated cytolysis.

The serious shortage of human organs available for transplantation has engendered a heightened interest in the use of animal organs (xenografts) for transplantation.However, the major barrier to successful discordant xenogeneic organ transplantation is the phenomenon of hyperacute rejection. Hyperacute rejection results from the deposition of high-titer preformed antibodies that activate serum complement on the luminal surface of the vascular endothelium, leading to vessel occlusion and graft failure within minutes to hours. Although endogenous membrane-associated complement inhibitors normally protect endothelial cells from autologous complement, they are species restricted and thus confer limited resistance to activated xenogeneic complement. To address the pathogenesis of hyperacute rejection in xenotransplantation, transgenic mice and a transgenic pig were engineered to express the human terminal complement inhibitor hCD59. High-level cell surface expression of hCD59 was achieved in a variety of murine and porcine cell types, most importantly on both large vessel and capillary endothelium.hCD59-expressing porcine cells were signicantly resistant to challenge with high-titer anti-porcine antibody and human complement. These experiments demonstrate a strategy for developing a pig-to-primate xenogeneic transplantation model to test whether the expression ofa human complement inhibitor in transgenic pigs could render xenogeneic organs resistant to hyperacute rejection.

SummaryType C retroviruses endogenous to various nonprimate species can infect human cells in vitro, yet the transmission of these viruses to humans is restricted. This has been attributed to direct binding of the complement component C lq to the viral envelope protein p15E, which leads to classical pathway-mediated virolysis in human serum. Here we report a novel mechanism of complement-mediated type C retrovirus inactivation that is initiated by the binding of"natural antibody" [Ab] (anti-o~-galactosyl Ab) to the carbohydrate epitope Galod-3Gal[31-4GlcNAc-R. expressed on the retroviral envelope. Complement-mediated inactivation of amphotropic retroviral particles was found to be restricted to human and other Old World primate sera, which parallels the presence ofanti-0t-galactosyl natural Ab. Blockade or depletion ofanti-o~-galactosyl Ab in human serum prevented inactivation of both amphotropic and ecotropic murine retroviruses. Similarly, retrovirus was not killed by New World primate serum except in the presence of exogenous anti--~-galactosyl Ab. Enzyme-linked immunosorbent assays revealed that the cx-galactosyl epitope was expressed on the surface of amphotropic and ecotropic retroviruses, and Western blot analysis further localized this epitope to the retroviral envelope glycoprotein gp70. Finally, down-regulation of this epitope on the surface of murine retroviral particle producer cells rendered them, as well as the particles liberated from these cells, resistant to inactivation by human serum complement. Our data suggest that anti-~-galactosyl Ab may provide a barrier for the horizontal transmission of retrovirus from species that express the a-galactosyl epitope to humans and to other Old World primates. Further, these data provide a mechanism for the generation of complement-resistant retroviral vectors for in vivo gene therapy applications where exposure to human complement is unavoidable.